title: A rice virescent-yellow leaf mutant reveals new insights into the role and assembly of plastid caseinolytic protease in higher plants.
abstract: The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS). The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana). In this work, we isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period. The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.
authors.affiliation: National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Dong
authors.forename: Gui-Lin
authors.initials: GL
authors.lastname: Fei
authors.forename: Chuan-Yin
authors.initials: CY
authors.lastname: Wu
authors.forename: Fu-Qing
authors.initials: FQ
authors.lastname: Wu
authors.forename: Yu-Ying
authors.initials: YY
authors.lastname: Sun
authors.forename: Ming-Jiang
authors.initials: MJ
authors.lastname: Chen
authors.forename: Yu-Long
authors.initials: YL
authors.lastname: Ren
authors.forename: Kun-Neng
authors.initials: KN
authors.lastname: Zhou
authors.forename: Zhi-Jun
authors.initials: ZJ
authors.lastname: Cheng
authors.forename: Jiu-Lin
authors.initials: JL
authors.lastname: Wang
authors.forename: Ling
authors.initials: L
authors.lastname: Jiang
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.forename: Xiu-Ping
authors.initials: XP
authors.lastname: Guo
authors.forename: Cai-Lin
authors.initials: CL
authors.lastname: Lei
authors.forename: Ning
authors.initials: N
authors.lastname: Su
authors.forename: Haiyang
authors.initials: H
authors.lastname: Wang
authors.forename: Jian-Min
authors.initials: JM
authors.lastname: Wan
mesh_terms: D001665:Binding Sites; D002734:Chlorophyll / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000235:genetics; D003001:Cloning, Molecular; D049071:Endopeptidase Clp / Q000235:genetics* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D012275:Oryza / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D010788:Photosynthesis / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D018087:Plastids / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping; D020798:Two-Hybrid System Techniques
chemical_list: D010940:Plant Proteins; D002734:Chlorophyll; D049071:Endopeptidase Clp